The complement system is part of the innate immune system, which protects the body against invading pathogens. Initiator molecules of complement system recognize conserved patterns on the surface of microorganisms, which then initiates a cascade of enzymatic reactions. This results in targeted removal and in some cases destruction of the microorganisms. Three activation pathways of the complement system have been described; the classical pathway, the alternative pathway and the lectin pathway. C1q activates the classical pathway; the alternative pathway is activated by spontaneous hydrolysis of C3 and in some cases properdin. The lectin pathway is activated by mannose-binding lectin (MBL) and the Ficolins (Ficolin-1, Ficolin-2 and Ficolin-3). Ficolin-1 is expressed by cells of myeloid origin and is found circulating in serum at low concentrations. Ficolin-2 and Ficolin-3 are both expressed in the liver and are circulating in serum at a mean concentration of 5 μg/ml and 24 μg/ml, respectively. Furthermore, Ficolin-3 is abundantly expressed in the lungs, suggesting a role for this molecule in pulmonary immune defence.
In humans, three ficolin genes have been identified: FCN1, FCN2 and FCN3, which encode Ficolin-1 (synonymous with M-ficolin and Ficolin/P35-related protein), Ficolin-2 (synonymous with L-ficolin, Ficolin/P35 and Hucolin) and Ficolin-3 (synonymous with H-ficolin, Hakata antigen and thermolabile β2-macroglycoprotein, HAKA1, collagen/fibinogen domain-containing lectin 3 P35), respectively. FCN1 and FCN2 are both located on chromosome 9q34, and are 80% homologous at the amino acid level, whereas FCN3 is assigned to chromosome 1 (1p36.11, 1p35.3) ((Endo, Y., et al. (1996), Sugimoto, R. et al., (1998)).
On the amino acid level, Ficolin-3 reveals ˜40% homology with both Ficolin-1 and Ficolin-2. FCN1 contains nine exons, whereas FCN2 and FCN3 are composed of eight exons. Ficolin-2 and Ficolin-3 are found in serum and exhibits inter-individual variation in serum concentrations (Kilpatrick, D. C. et al., (1999), Matsushita, M. et al., (1996), Yae Y., et al., (1991)). FCN2 is predominantly expressed in the liver and FCN3 in the liver and lung (Endo, Y., et al. (1996), Akaiwa, M., et al., (1999)). Ficolin-1 is expressed in the lung, the spleen and by undifferentiated monocytes (Endo, Y., et al. (1996), Harumiya, S., et al., (1996), Lu, J., et al., (1996)). Ficolin-1 is present in serum in lower concentrations (Honore, C., et al (2008)).
The ficolins are synthesized as a single polypeptide containing N-terminal collagen-like and C-terminal fibrinogen-like sugar-binding domains, which are oligomerized into higher oligomeric forms comprising triple helix structures (Matsushita, M., Fujita, T. (2001)). The collagen-like multimeric structure is shared with C1q, mannose-binding lectin (MBL) and surfactants proteins A and D (SP-A and SP-D).
The fibrinogen-like domains of Ficolin-1 and Ficolin-2 have been shown to interact with carbohydrates, such as N-acetylglucosamine (GlcNAc) (Teh, C., et al., (2000)).
Moreover, a general specificity for N-acetylated groups for Ficolin-2 has also been demonstrated (Krarup, A., et al., (2004)). Both Ficolin-1 and Ficolin-2 appear to bind to different types of bacteria and Ficolin-2 may specifically bind to lipoteichoic acid from gram-positive bacteria (Teh, C., et al., (2000), Lynch, N. J., et al., (2004)). The ligands for Ficolin-3 are unknown, but distinct binding to certain strains of bacteria has been demonstrated (Tsujimura, M., et al., (2002)). Ficolin-1 has been shown to enhance uptake of bacteria to monocytes and Ficolin-2 and Ficolin-3 have been shown to interact with the Ficolin-3-associated serine proteases enabling activation of the complement system (Teh, C., et al., (2000), Matsushita, M., et al., (2000), Matsushita, M., et al., (2002)). Taken together, these results provide evidence for the fact that the ficolins are important molecules in imparting innate immunity.
Conventional functional assays for evaluating complement activation through the different pathways mostly use an MBL-specific ligand for assessing the lectin pathway. WO/2005/051662 describes methods for detecting Ficolin-2 dependent activation of the lectin pathway.
Recently a frameshift mutation (FCN3+1637delC) in the gene (FCN3) encoding Ficolin-3 was described (Hummelshoj, T., et al (2005)). This mutation results in premature termination leading to expression of a non-functional protein. Individuals heterozygous for the FCN3+1637delC mutation showed significantly lower levels of Ficolin-3 in serum and from studies using recombinant protein expression it was found that an individual homozygous for this mutation would result in Ficolin-3 deficiency (Munthe-Fog, L., et al (2008)).
The allele frequency of this mutation was 0.011 in Caucasians, meaning that homozygosity for the FCN3+1637delC mutation will be found in approximately 1 out of 8000-10, 000 individuals. The frameshift mutation (FCN3+1637delC) was analysed in are large group of patients with suspected non-HIV related primary immunodeficiencies. We identified one patient that was homozygous for the FCN3+1637delC allele. This patient suffered from unexplained recurrent bacterial lung infections, brochiectasis, lung fibrosis and obstructive lung disease and had experienced one incidence of cerebral abscesses. Thus, Ficolin-3 deficiency appears to result in a novel complement deficiency state. This observation calls for an assay that can identify functional and genetic deficiencies of Ficolin-3.